Many peptides, particularly peptides from about three to about 20 amino acids in length, are unstable in low concentrations in aqueous formulation and tend to lose biological activity, although retaining chemical stability and structure. There also exist a number of larger peptides or polypeptides which experience a similar loss of activity upon the use of low dose aqueous concentrations, e.g., encephalin, tuftsin, interleukin 2, and the brain neuropeptide, Substance P. At present the only known way to preserve the biological activity of such peptides or proteins in aqueous formulations appropriate for injectable clinical dosages is storage in high concentrations. Generally, high concentrations for storage greater than 50 mg/mL require strict refrigeration conditions, e.g., between 2.degree. to 8.degree. C., or approximately 4.degree. C. Smaller concentrations, e.g., less than 10 mg/mL are frequently unstable even under strict refrigeration conditions.
One example of a peptide which experiences such biological activity loss at low concentrations in aqueous formulations is thymopentin, a pentapeptide of proven pharmacological use and significance. See, U.S. Pat. No. 4,190,646 and Goldstein, G. Nature (London) 247: 11-14 (1974); Basch, R.S. and Goldstein, G., Proc. Natl. Acad. Sci. U.S.A., 71: 1474-1478 (1974); Scheid, M. P. et al, J. Exp. Med., 147: 1727-1743 (1978); Scheid, M. P. et al, Science, 190: 1211-1213 (1975); Ranges, G. E. et al, J. Exp. Med., 156: 1057-1064 (1982); T. Audhya et al., Biochem, 20: 6195-6200 (1981); Venkatasubramanian, K. et al, Proc. Natl. Acad. Sci. U.S.A., 83: 3171-3174 (1986); Malaise M. G. et al, in "Immunoregulatory UCLA Symposium on Molecular and Cellular Biology", eds. Goldstein, G., et al (Liss, New York) (1986); Sunshine, G. H. et al, J. Immunol., 120: 1594-1599 (1978) and E. Rentz et al, Arch. Geschwulstforsch, 54(2): 113-118 (1948). See also U.S. Pat. Nos. 4,261,886; 4,361,673; 4,420,424; and 4,629,723. These references and patents are incorporated by reference herein for their disclosure of thymopentin and methods for its preparation.
In experiments with thymopentin, it was observed that the optimal doses of thymopentin in an injectable aqueous formulation for immunostimulation in laboratory animals was about 1 .mu.g/kg of body weight. For laboratory use, these dosages were prepared immediately prior to use. Subsequent human clinical research indicated that much larger doses, approximately 1 mg/kg body weight or greater, were required to produce a clinical pharmacological result in the patient. Preparations of clinical quantities of thymopentin at a dosage of less than 10 mg/mL in aqueous formulations were frequently found to be biologically inactive, thus accounting for a lack of efficacy of these lower concentration formulations in clinical studies.
Such activity loss in a peptide for pharmaceutical use may effect the therapeutic treatment of a patient requiring a particular pharmacologically active peptide. A loss of activity in the dosage unit will result in too little active peptide being delivered to the patient in the normal dosage unit. Thus, the appropriately effective dose of the peptide will not be given to the patient. If the activity loss is less than complete, such a variable loss will render it impossible for a practical pharmaceutical dosage to be accurately determined. Simply raising the dosage level of the peptide to compensate for this loss is not practical because the degree of loss would be unknown and excess dosages of most pharmaceuticals carry an increased risk of serious side effects. Such inefficient methods to compensate for activity loss of the peptide will also increase the cost of the pharmaceutical in question.
Additionally refrigeration at between approximately 2.degree.-8.degree. C. is required to maintain the biological activity of certain peptides and polypeptides, including thymopentin. For example, concentrations of thymopentin of 50 mg/mL require refrigeration to retain activity for up to and after two years, the conventional industry standard for stable pharmaceutical preparations. Lower concentrations often do not retain activity even with refrigeration. This need to refrigerate these peptides in pharmaceutical dosages severely hampers the manufacture, storage, transport and use of such peptides. For example, lack of refrigeration or fluctuation in refrigeration temperature during transportation of such peptides for therapeutic or other uses or during storage of such peptides, e.g., in countries where the appropriate clinical and pharmaceutical facilities are not available, may prevent the effective use of such peptides, even in higher concentrations.
Therefore a need exists in the art for methods of preparing an aqueous formulation of thymopentin in a manner which will retain the biological activity of clinical quantities thereof.